Fluorescent energy transfer is one mechanism which has been proposed for use in biosensor applications. In selecting optimum donor-acceptor dye pairs a number of the following criteria should be met: 1) low overlap between the absorption spectra of donor and acceptor so that the direct excitation of the acceptor by the laser line is minimal: 2) high overlap between the emission spectra of donor and absorption spectra of acceptor so that the energy transfer efficiency is maximal; 3) good separation between the emission maxima of the donor and acceptor so that the ratio of the two intensities can be taken; 4) the donor should be able to be excited with a laser; 5) the fluorescence maxima of both donor and acceptor should be at a wavelength higher than serum fluorescence; 6) both donor and acceptor should have high extinction coefficients and high fluorescence quantum yields to ensure maximum sensitivity.
In order to select optimum donor-acceptor pairs and properly characterize their fluorescence properties various aspects of the pairs and their interactions with proteins (e.g., antibodies and antigens) need to be considered including such issues as: 1) energy transfer properties in solution; 2) spectral separation to determine the energy transfer efficiency; 3) other interactions between dyes besides energy transfer; 4) better methods to determine the degree of labeling; 5) calculation of the characteristic distance for all the potential donor-acceptor pairs; 6) fluorescence lifetimes of individual donors and acceptors, as well as the donor-acceptor pairs.
It is deemed important to select respective donor and acceptor dyes on the basis of maximum energy transfer efficiency. Clearly, accurate and relatively simple analytical techniques for calculating such efficiency values would be of interest to persons using fluorescent energy transfer techniques would be of value.
Energy transfer efficiency can be calculated from either spectral or lifetime measurements. Since spectral measurements are preferred for biosensor applications, methods are needed to calculate transfer efficiency based on the spectral data. Based on the quenching of donor fluorescence, the transfer efficiency is given by: ##EQU1## where, E is energy transfer efficiency; F.sub.d is the fluorescence intensity of donor in the absence of acceptor; A.sub.d is the absorbance of donor in the absence of acceptor; F.sup.a.sub.d is the fluorescence intensity of donor in the presence of acceptor; A.sup.a.sub.d is the absorbance of donor in the presence of acceptor.
In order to calculate E, both the fluorescence and absorption spectrum of the D-A-IgG complex have to be separated into components of D-IgG and A-IgG. A curve fitting method using Gaussian distribution functions was one technique attempted to perform such a separation. It was found, however, that at least three Gaussian distributions were required to fit even a single emission peak. One would have to fit six Gaussian functions for a simple two-component system. Clearly this method was too complicated to be of any practical value.
R. B. Gennis et al. in Biochemistry, Vol. 11, No. 13, 1972, 2509-2517 mention the following ways in which transfer efficiencies might be calculated: (1) numerical integration; (2) an equation, as depicted in its Appendix, involving two donors and one acceptor; and (3) Markov chain methods.